Neonatal Neutrophil Inflammatory Responses: ParallelStudies of Light Scattering, Cell Polarization,

Chemotaxis, Superoxide Release, andBactericidal Activity

B. Wolach,* D. Sonnenschein, R. Gavrieli, O. Chomsky, A. Pomeranz, and I. YuliDepartment of Pediatrics, The Pediatric Hematology Unit and The Laboratory for Leukocyte Functions, Meir General Hospital, Sapir

Medical Center, Kfar Saba, Israel and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Neutrophil dysfunction among newborn infants, especially those born prematurely, iswell recognized, but the mechanism responsible for this phenomenon is yet to be clari-fied. In this study, we evaluated the stimulus response coupling in neutrophils from 90healthy newborns and 96 healthy adults in an effort to establish whether defective neo-natal neutrophil function is a result of impaired signal perception or immature respon-siveness. Measurement of rapid- and slow-light scattering responses (LSR) to 1 µM FMLPstimulation revealed that neonatal neutrophils have about one-half the correspondingresponsiveness of adult cells (rapid-LSR: 6.1 ± 3.1 arbitrary light intensity units vs. 12.0± 2.8, P < .001; and slow-LSR: 5.0 ± 2.5 vs. 9.1 ± 2.0; P < .001). The same markedly reducedactivity was observed in newborn neutrophil chemotaxis and bactericidal activity in com-parison with adult cells. Nevertheless, low FMLP concentrations (less than 1 nM) inducedno difference in cell polarization between newborn and adult neutrophils, yet at higherFMLP concentrations, the newborn revealed significantly reduced cell polarization. Ourdata suggest that newborn infants bear a fully functional FMLP signal perception but lackthe full capacity of inflammatory responsiveness. Am. J. Hematol. 58:8–15, 1998.© 1998 Wiley-Liss, Inc.

Key words: bactericidal activity; cell polarization; light scattering responses; neonatalneutrophil chemotaxis; neutrophil dysfunction

INTRODUCTION

Newborn infants, particularly those born prematurely,are more likely to develop severe pyogenic infectionsthan older infants or adults [1,2]. This predilection prob-ably results from humoral, phagocytic, and/or cellularimmunological deficiencies in the neonate [3,4]. Leuko-cyte dysfunction [5–7] including poor chemotaxis [8–10], defective adherence and aggregation [11–13], andinsufficient opsonization have been reported to occur inthe neonatal period [3,4,14–16]. The causes for the leu-kocyte defects have not yet been fully resolved, but mayreflect, among other causes, an incompetent cellular ap-paratus [17,18] or inadequate signal perception.

The density of the receptors for N-formyl-L-methionyl-Leucyl-L-phenylalanine (FMLP) on the neo-natal neutrophil surface has been reported to be compa-rable to that found on adult neutrophils [19]. However,

membrane fluidity, which is known to regulate signalperception [20,21], has been found to be increased inneonatal neutrophils [22], and as a consequence leads toblunted neutrophil responses.

Measurement of light scattering [23], which includesreading both the incident and perpendicular responses,has proven itself to be unique in that it is capable ofassessing the very early neutrophil response to chemoat-tractant stimulation, since it monitors the initial mem-brane and cytoskeleton rearrangements following cellstimulation. Furthermore, this assay has the advantage of

*Correspondence to: B. Wolach, Department of Pediatrics, Meir Gen-eral Hospital, 44281 Kfar Saba, Israel.

Received for publication 6 February 1997; Accepted 12 November1997

American Journal of Hematology 58:8–15 (1998)

© 1998 Wiley-Liss, Inc.

distinguishing between inflammatory signals that areperceived by the single FMLP receptor, and are differ-entially directed to either chemotaxis or bactericidal re-sponse pathways. The latter quality enables us to estab-lish whether the response inhibition is due to impairedcell machinery mechanisms (e.g., motility, secretory oroxygen burst mechanisms), or to blunted receptor per-formance. As far as we know, the perpendicular LSR hasnot yet been applied to the study of neonatal neutrophilresponses. Hence, by using this particular LSR method-ology, we expected to ascertain whether the deficientinflammatory responses of neonatal neutrophils could beattributed to the receptor signal perception or to thedownstream cellular response-apparatus.

PATIENTS AND METHODSSubjects

Ninety newborn infants were included in variousstages of this study. All were full-term infants born vagi-nally to healthy mothers after normal pregnancy and de-livery. Their birth weight ranged between 2,800–3,400 g,and their Apgar scores were 9 and 10 at 1 and 5 min,respectively. Blood samples were taken at 2–5 days afterbirth. The study was approved by the Helsinki Commit-tee at the Meir General Hospital, Sapir Medical Center,Kfar Saba, Israel. A control adult study group consistedof 96 healthy volunteers aged 20–45 years.

Reagents

N-formyl-L-methionyl-L-leucyl-L-phenylalanine(FMLP), Phorbol myristate acetate (PMA), N-2-hydroxyethylpiperazine-N8-2-ethanesulfonic acid(HEPES), superoxide dismutase, ferricytochrome-C, bo-vine serum albumin, Krebs-Ringer phosphate buffer(KRP), and phosphate buffer saline (PBS) were pur-chased from Sigma Chemical Co. (St. Louis, MO). Ear-le’s salt solution supplement with L-glutamine (M199medium) was purchased from Biological Industries Inc.(Kibbutz Beit Haemek, Israel). Hank’s Balanced Salt So-lution (HBSS) was prepared by the Weizmann InstituteBiological Services. Dextran T-250 was purchased fromPharmacia Fine Chemicals (Pharmacia Laboratories,Pitscataway, NJ). Heparin (5,000 U/ml) was purchasedfrom LEO Ltd. (Ballerup, Denmark).

Polymorphonuclear Leukocyte(Neutrophil) Isolation

Five milliliters of heparinized peripheral blood fromnewborns, or 20 ml from adults, was mixed with equiva-lent volumes of 3% dextran (T-250) in saline. Neutro-phils were isolated according to Boyum [24], and re-sidual erythrocytes were removed by hypotonic lysis.The neutrophils were then suspended in HBSS supple-mented with 10 mM HEPES (HHB), pH 7.4, for light

scattering assays, in M199 medium for chemotaxis as-says, in Krebs-Ringer buffer (KRP) for superoxide anionrelease assays, and in PBS supplemented with 0.2% D-glucose and 1% bovine serum albumin (PBS-GA) forbactericidal activity assays.

Morphological Response

Neutrophil suspensions at 4 × 106 cells in 0.4 ml HHBwere vigorously stirred (500 rpm) at 37°C. The neutro-phils were stimulated by adding 4ml of FMLP-solutionto achieve final concentrations of 10 pM, 100 pM, 1 nM,10 nM, 100 nM, and 1mM. Kinetic studies revealed thatthe cell polarization is fully apparent 30 sec after stimu-lation. Cell morphology was captured by the addition of0.4 ml of ice-cold 10% formaldehyde in HHB, and clas-sified by light microscopy as to whether there werespherical or polar-shaped cells. The percentage of polar-ized cells in triplicate samples of 500 cells each wasdetermined.

Light Scattering Responses

Incident and perpendicular light scattering responsesof neutrophils were measured in a dual aggregation meter(DP-247E; Sienco Inc., Morrison, CO), as previously de-scribed [23,25]. Briefly, neutrophil samples of 0.4 ml at107 cells/ml HHB were suspended in 1-ml cuvettes at37°C. Baseline levels of light scattering from neutrophilsuspensions were stabilized prior to stimulation by fineadjustment of the cell stirring in the range of 500 ± 50rpm. Stimulation was achieved by injecting 4mL of ei-ther 10 nM or 1mM FMLP. Figure 1 depicts typicalpatterns of the incident and perpendicular responses. Theresponse of the incident light (lower tracing) is charac-terized primarily by one relatively slow peak, on theorder of 5 min. This effect is quantified by measuring itspeak amplitude or the area under the tracing integratedfrom the time of stimulation to 3 min (integration per-formed with the aid of the Auto Cad package). The re-sponse, as detected by perpendicular scattering (uppertracing), is comprised of two transient peaks. The first,the rapid-light scattering response (rapid LSR), is definedby the 10 ± 1 sec required from stimulation to peakreduction in light intensity, and the additional 10 sec forthe restoration of light intensity [23]. The ensuring slow-light scattering response (slow-LSR) yields a peak afterabout 50 ± 10 sec from stimulation, followed by a ratherslow return to pre-stimulated light scattering level, in theorder of several minutes. The area above the perpendicu-lar light scattering was integrated for 3 min after stimu-lation. The possibility of mixing artefacts was negated atthe introduction of the light-scattering method [23,25].This was proven by using cell-free systems, and non-stimulants (buffers). In addition, responses to stimulationwith a spectrum of six orders of magnitude of several

Neonate Neutrophil Inflammatory Response 9

chemoattractants yielded the exact 10 sec peak in everyresponse.

Chemotaxis Assay

A 48-well chemotactic microchamber (Neuro Probe,Inc. Bethesda, MD) was used to determine random mi-gration and chemotaxis [26]. M199 medium or FMLP ata concentration of 10mM was added to the wells of thebottom plate. A polycarbonate filter sheet, PVP-Free,containing 3-mm pores (Nucleopore Corp., Pleasanton,CA) was placed on top of the bottom plate. A matchedupper plate was attached to the bottom one and fixed

firmly. Fifty microliters containing 5 × 104 neutrophils(i.e., 106 cells/ml) was placed in upper wells. The cham-ber was incubated for 60 min in a humidified atmosphereat 37°C. After incubation, the cells from the upper side ofthe filter were wiped off, and the filter sheet was stainedwith May-Grumwald-Giemsa. The number of migratingcells was determined in nine fields by light microscopy(×40 objective and a ×10 ocular equipped with a finegrid). Net chemotaxis was calculated by subtracting therandom migration (M199 medium in the bottom wells)from the FMLP-driven neutrophils. Experiments werecarried out in duplicate.

Bactericidal Activity

The maximal bactericidal activity was evaluated aspreviously reported [27]. Bacteria (Escherichia coli)were freshly harvested prior to each experiment, and al-lowed to enter an early stationary growth phase (18 hr at37°C). The final density of the bacteria was assessed byspectrophotometry at 590 nm. A suspension of neutro-phils at 5 × 106 cells/mL PBS-GA (final density) wasincubated with bacteria at a neutrophil bacteria densityratio of 1:5 or 1:10, respectively, in the presence of 10%autologous serum. The incubating systems were agitatedgently for 30 or 90 min in a 37°C chamber. After incu-bation, the neutrophils were lysed with 4 volumes ofdistilled water, plated (30 mL) in triplicate in broth agarplates, and incubated for 24 hr at 37°C. Each experimentincluded two controls that were comprised of PBS-GAand bacteria or autologous serum and bacteria. In theseexperiments, we observed less than 0.02 log decrease inbactericidal activity (n > 100) in the control chambers.

Superoxide Anion Release

The assay was carried out as previously reported [28].Neutrophils at a final density of 5 × 105 cells/mL KRPwere preincubated with or without 214 U/mL superoxidedismutase for 5 min at 37°C. Then, 60mM ferricyto-chrome-C was added, and the response was initiated bythe prompt addition of 0.1mM FMLP or 1 mg/mL phor-bol myristate acetate. The reaction mixtures were incu-bated for 10 min at 37°C. The reactions were stopped byplacing the tubes in melting ice. The neutrophils werecentrifuged (500g for 10 min at 4°C), and the opticaldensity of the supernatant was determined at 550 nm.The results of triplicate determinations were averaged,and superoxide anion release was calculated according toMassey, using the extintion coefficient of 21,000 M−1

cm−1.

Statistical Analysis

Both the Student’st-test and the Mann-Whitney non-parametric test were used for data analysis of all leuko-cyte functions. In addition, the Pearson correlation coef-

Fig. 1. Light scattering response of a neutrophil suspen-sion to a single stimulation with 10 nM FMLP, which wasachieved by an injection at the zero time point (indicated bythe arrows). The response is characterized by a single inci-dent light wave (peaks at approximately 1 min; bottom), andtwo sequential low peaks (10 and 40 sec, respectively) ofperpendicular scattered light (top). The amplitudes of theresponses are depicted by the vertical bars, and the inte-grations of either the incident or the perpendicular re-sponses of the first 3 min are indicated by the respectiveshaded areas.

10 Wolach et al.

ficient was used for assessing statistical correlations oflight scattering, chemotaxis, and bactericidal activity.

RESULTS

The response of neonatal neutrophils to FMLP-stimulation was found to be identical to that of the adultneutrophils with respect to timing and amplitude-ratio(i.e., rapid-LSR/slow-LSR, see Fig. 2). The peak timingof the rapid-LSR and the slow-LSR of both neonate andadult neutrophils was strictly comparable: at any FMLPstimulation dose, the rapid-LSR reached its peak effect at10.5 ± 0.9 sec (n4 22 newborn neutrophils), and at 10.7± 1.0 sec (n4 9 adult cells) after stimulation, while theslow-light scattering responses reached their peaks at 61± 15 and 59 ± 10.5 sec, respectively. Stimulation by 10nM FMLP, the optimal dose for chemotaxis induction,yielded rapid- to slow-LSR amplitude ratios of 1.7 ± 0.5and 1.9 ± 0.5 for newborn infant and adult neutrophils,respectively. Also, stimulation with 1mM FMLP, theoptimal dose for induction of bactericidal activities,

yielded the same rapid- to slow-LSR amplitude ratio, i.e.,1.3 ± 0.2, for newborn and adult cells. These findingsindicate that, when compared with adult cells, newbornneutrophils respond properly to FMLP stimulation, asdetermined by the LSR method [23].

Quantification of neutrophil responsiveness to FMLPstimulation revealed differences between neonates andadults. The amplitude of and the area under the light-output traces (see Fig. 2 and Table I) of neonatal neutro-phils were about one half that of the adult cells for boththe rapid and slow LSR. These differences reached highstatistical significance in the rapid-LSR after stimulationby the either 10 nM or 1mM FMLP. The same highstatistical significance was observed in the slow-LSR atthe optimal bactericidal activity dose of 1mM FMLP. Incontrast, there were no differences between adult andneonatal neutrophils in the incident light-scattering re-sponse, either in amplitude or in the area under the re-sponse curve.

To relate the light scattering results to standard cellularand biochemical assays, we tested neutrophil polariza-tion, chemotaxis and bactericidal activities. Figure 3 de-picts the FMLP dose-dependent polarization of adult andnewborn neutrophils. As previously observed [29], neu-trophil polarization reached its peak at 10 nM FMLP,with some decline in the percentage of polarized cells athigher doses. We observed that polarization of adult andnewborn neutrophils was practically indistinguishable atconcentrations of FMLP lower than 1 nM. At higherconcentrations, however, newborn neutrophils revealedsignificantly less cell polarization.

Chemotaxis was found to generally follow the obser-vations in the cell polarization experiments: chemotaxisof newborn neutrophils was only one half that of adultcells (Table II). However, since the random migration ofboth adult and neonatal cells was almost identical, netchemotaxis of the newborn neutrophils was only one-third of that of adult cells (see Table II). Similarly, thebactericidal activity of the newborn neutrophils reachedone-third the bactericidal capacity of adult cells (TableIII, at 90 min).

Significant statistical correlation was found betweenthe rapid-LSR and neutrophil chemotaxis (P < 0.01), andslow-LSR and bactericidal activity (P < 0.01).

Nevertheless, as previously reported [19], we also ob-served that superoxide anion generation in response toFMLP (e.g., 0.1mM) of neonates exceeded that of theadult cells, i.e., 1.9 ± 0.7 (n4 10) and 1.4 ± 0.5 (n4 14)nmol/min/106 cells, respectively (P < 0.005). However,the 1 mg/mL PMA-stimulated superoxide production inadult and newborn cells was similar (i.e., 6.0 ± 2 (n 415) and 7.0 ± 1.4 (n4 24) nmol/min/106 cells, respec-tively).

Fig. 2. The perpendicular light scattering responses to 10nM FMLP (A,B) and 1 µM FMLP (C,D) of adult neutrophilsuspensions (A,C) and newborn infant cell suspensions(B,D).

Neonate Neutrophil Inflammatory Response 11

DISCUSSION

Neutrophils exhibit a unique behavioral pattern uponstimulation that is manifested in the rapid modulation oftheir light scattering properties. The perpendicular LSR,in particular, might even distinguish between the earlychemotaxis and late inflammatory responses [20,21].

Hence the short and transient characteristic of the rapid-LSR qualifies it as an indicator of chemoattractant signalperception, whereas the slow-LSR indicates degranula-tion and bactericidal activity.

Although there is still little experimental data to ex-plain the phenomena, we speculate that the light effectsare the result of an extremely synchronous and massiverearrangement of organelles in the responding neutro-phils.

The light scattering effects are probably a result of twophenomena. The first being the enhanced turnover ofmicrofilaments with the close bundling of F-actin, whichthen appear as dark spots at the base of the pseudopodiaof polarizing neutrophils. This response by microfila-ments is one of the earliest and most fundamental neu-

TABLE I. Comparison of Neonatal (n = 22) and Adult (n = 9) Neutrophils’ Light Scattering Response to 10 nM and 1 µM FMLP*

a.10 nM FMLP 1mM FMLP

Measure (amplitude) Newborn infants Adults P value Newborn infants Adults P value

Rapid-LSR 5.6 ± 2.7 9.6 ± 2.2 <.001 6.1 ± 3.1 12.0 ± 2.8 <.001Slow-LSR 2.7 ± 2.5 5.2 ± 1.7 <.02 5.0 ± 2.5 9.1 ± 2.0 <.001Incident-LT 3.5 ± 1.9 3.6 ± 1.9 NS 9.6 ± 3.1 10.1 ± 4.7 NS

b.10 nM FMLP 1mM FMLP

Measure (area) Newborn infants Adults P value Newborn infants Adults P value

Slow-LSR 7.6 ± 3.1 12.1 ± 3.5 <.005 11.4 ± 5.3 20.4 ± 6.3 <.001Incident-LSR 7.5 ± 4.2 7.4 ± 3.6 NS 21.5 ± 6.7 23.3 ± 6.9 NS

*Amplitude of response (cm) shown ina; area under the curve (cm2) between stimulation and after 3 min shown inb (mean ± SD).

Fig. 3. Whole cell polarization of adult ( d) and newborn ( h) neutrophils in response to the indicated FMLP concentrations.The experimental points represent the mean and the standard deviation of triplicate samples, each of 500 cells, derivedfrom 10 adult and 10 newborn infant donors. The difference between the adult and the newborn cell polarization reachedhigh statistical significance at FMLP stimulation by 10 nM and higher ( P < 0.001).

TABLE II. Comparison Between the Neutrophil Migration ofNewborn Infants (n = 30) and Adult (n = 40) (Mean ± SD)

MeasureNewborn infants

(no. of cells)Adults

(no. of cells) P value

Chemotaxis migration 53 ± 18 108 ± 19 <.001Random migration 30 ± 14 34 ± 14 NSNet chemotaxis 25 ± 11 70 ± 14 <.001

12 Wolach et al.

trophil reactions to surface stimuli [30–34]. The secondphenomenon is the discharging of secreting granules oftheir protein-rich content [35,36].

The observed two-phase, timed response led us tospeculate that the rapid-LSR depicts the association ofthe F-actin fibers into matrix like structures. Actin as-sembly is generally mediated through G proteins [37,38].In addition, we speculate that the slow-LSR depicts thesecretion event, whereby the secretion involves mechani-cal movement of all organelles to the plasma membraneand their fusion. Since the secretion organelles are dis-persed in the entire cytoplasm, the probability that theywould reach the plasma membrane simultaneously islimited. It is, therefore, likely to be a slower and lesssynchronous event than the bundling of the F-actin fi-bers.

In this study, neutrophil functions, which are known tobe impaired in the neonatal period [3–5,7–13,16], wereanalyzed to gain a better understanding of the specificneonatal phagocytic impairment at the intracellular level.In addition, the state of the inflammatory responsemechanisms themselves was assessed by directly moni-toring the neutrophil chemotaxis, superoxide anion re-lease, and bactericidal activity.

Neonatal neutrophils demonstrated the proper qualita-tive FMLP-induced light scattering responses. This con-clusion is supported by the following: (1) the shape andtiming of the peaks of either the perpendicular rapid- orslow-LSR of neonatal cells were identical to those ob-tained with adult cells; (2) the light scattering responsesof neonatal neutrophils yielded amplitude ratios of rapid-to slow-LSR and slow LSR to incident-LT tracings thathighly corresponded to normal adult neutrophils.

Neonatal neutrophils did demonstrate quantitative ab-normalities in their FMLP-induced LSR when comparedwith adult cells, however. The magnitude of neonatalneutrophil responses reached approximately only one-half that of corresponding adult LSR, irrespective ofwhether the response was assessed by the ‘‘area underthe peak’’ parameter, or by direct recording of the am-plitude of peaks (Table I). In addition, while adult cellsdisplayed a relatively narrow variation in response am-plitudes, neonatal responses demonstrated greater vari-ability. The latter agrees with previous studies [10,22,39–41], and could be accounted for by the coexistence of

different sub-populations in neutrophils of neonates,which might relate to various states of activation and/ormaturation [29,40–42]. It is plausible to assume that thematuration of neonatal neutrophils during the postnatalperiod is quite variable.

Previous studies have shown that there is a monopha-sic increase in neonatal neutrophil LT, when stimulatedby either FMLP or C5a, which is commonly interpretedas slow and irreversible cell aggregation [12,13,43]. Inour study, the LT assay failed to distinguish betweenneonatal and adult neutrophils since we obtained similarincreasing LT response patterns for both cell types whenstimulated by FMLP at either 10 nM or 1mM. In con-trast, the two perpendicular scattering responses of thesame cell suspensions were markedly decreased in neo-natal cells. Within these responses, the rapid-LSR is cor-related with membrane ruffling [44] and whole-cell po-larization [29], while the slow-LSR is associated with thebactericidal activities of the neutrophils, and is physicallyaccounted for by the overall increase in the cellular re-fractive index following the lysosomal secretion [23,45].These results indicate that the interpretation of the LTresponse, which corresponds to the slow-LSR by doseresponse and kinetics, is less reliable than the perpen-dicular LSR, since it is composed of non-interacting,absorbed, and forward scattered-light. Furthermore, thecorrelation between increased LT and neutrophil aggre-gation has been already empirically invalidated [13,25].We, therefore, conclude that the neutrophil inflammatoryresponsiveness is preferably monitored by the perpen-dicular LSRs.

The cellular and biochemical responses of the neutro-phil to chemoattractant stimulation also revealed that thenewborn neutrophils bear approximately only half thecapacity of their counterpart adult cells. Reduced cellpolarization and chemotaxis corresponded to the partialrapid-LSR, and the reduced lysosomal secretion and bac-tericidal activity ran parallel to the restrained slow-LSR[23]. However, superoxide anion production reachedcomparable levels in both newborn and adult cells, afterstimulation by either the receptor-mediated FMLP, or themembrane-soluble PMA agent. This latter observation issufficient to suggest that the FMLP signal perception atthe receptor level of newborn infants is not different fromthat of adult cells.

TABLE III. Comparison Between the Neutrophil Bactericidal Activity of Newborn Infants (n = 23) and Adults (n = 23)(Mean ± SD)

Incubation 30 min (decrease of colonies) Incubation 90 min (decrease of colonies)

Cell ratioa Newborn infants Adults P value Newborn infants Adults P value

1:5 0.45 ± 0.10 1.9 ± 0.6 <.001 0.75 ± 0.33 2.4 ± 0.6 <.0011:10 0.53 ± 0.12 1.7 ± 0.5 <.001 0.70 ± 0.26 2.2 ± 0.6 <.001

aCell ratio4 neutrophils:E. coli. Decrease in no. of colonies is derived by the following formula: Log (number ofE. coli colonies without neutrophils/numberE. coli colonies with neutrophils).

Neonate Neutrophil Inflammatory Response 13

Whole cell polarization of neonatal and adult neutro-phils was similar when stimulated by low FMLP concen-trations. Above 1 nM FMLP (optimal dose for chemo-taxis stimulation), the neonatal cell polarization could notmatch the extent of the adult response. These observa-tions suggest that neonatal neutrophils can respond toFMLP stimulation as much as adult cells, but their cy-toskeletal framework fails to comply with the demandsof a strong signal (i.e., higher than 10 nM FMLP). Itshould be noted that all inflammatory responses, whichhave been found to be impaired in the neonatal neutro-phils, involve rearrangement of the cytoskeleton to onedegree or another. Cytoskeleton rearrangement plays adirect role in cell polarization, chemotaxis, phagocytosis,and granule release (fusion with the plasma membraneafter removal of the actin barrier). It should be stressedthat the only function that appears to be fully active inneonatal neutrophils is cytoskeleton-free membrane as-sociate-superoxide production.

ACKNOWLEDGMENTS

We thank Dr. Jacob Nusbacher for valuable discus-sions and advice during the preparation of this manu-script.

REFERENCES

1. Wilson CB: Developmental immunology and role of host defenses inneonatal susceptibility. In Remington JS, Klein JO (eds): ‘‘InfectiousDiseases of the Fetus and Newborn Infants.’’ Philadelphia: WB Saun-ders, 1990, p 17.

2. Wolach B: Neonatal sepsis: Pathogenesis and supportive therapy.Semin Perinatol 21:28, 1997.

3. Hill HR: Host defenses in the neonate: Prospects for enhancement.Semin Perinatol 9:2, 1985.

4. Wilson CB: Immunologic basis for increased susceptibility of the neo-nate to infection. J Pediatr 108:1, 1986.

5. Hill HR: Biochemical, structural and functional abnormalities of poly-morphonuclear leukocytes in the neonate. Pediatr Res 22:375, 1987.

6. Mease AD: Tissue neutropenia: The newborn neutrophil in perspec-tive. J Perinatol 10:55, 1990.

7. Gahr M, Blanke R, Speer CP: Polymorphonuclear leukocyte functionin term and preterm newborn infants. Biol Neonate 48:15, 1985.

8. Sacchi F, Rondini G, Mingrat G, Stronati M, Gancia GP, MarsegliaGL, Siccardi AG: Different maturation of neutrophil chemotaxis interm and preterm newborn infants. J Pediatr 101:273, 1982.

9. Anderson DC, Hughes BJ, Smith CW: Abnormal mobility of neonatalpolymorphonuclear leukocytes. Relationship to impaired redistributionof surface adhesion sites by chemotactic factor or colchicine. J ClinInvest 68:836, 1981.

10. Anderson DC, Hughes BJ, Wible LJ, Perry GJ, Smith CW, BrinkleyBR: Impaired mobility of neonatal PMN leukocytes: Relationship toabnormalities of cell orientation and assembly of microtubules in che-motactic gradients. J Leukocyte Biol 36:1, 1984.

11. Krause PJ, Maderazo EG, Scroggs M: Abnormalities of neutrophiladherence in newborns. Pediatrics 69:184, 1982.

12. Olson TA, Ruymann FB, Cook BA, Burgess DP, Henson SA, ThomasPJ: Newborn polymorphonuclear leukocyte aggregation: A study of

physical properties and ultrastructure using chemotactic peptides. Pe-diatr Res 12:993, 1983.

13. Rochon YP, Frojmovic MM, Mills EL: Comparative studies of micro-scopically determined aggregation, degranulation and light transmis-sion after chemotactic activation of adult and newborn neutrophils.Blood 75:2053, 1990.

14. Wolach B, Dolfin T, Regev R, Gilboa S, Schlesinger M: The devel-opment of the complement system after 28 weeks’ gestation. ActaPaediatr 86:523, 1997.

15. Notarangelo LD, Chirico G, Chiara A, Colombo A, Rondini G, PlebaniA, Martini A, Ugazio AG: Activity of classical and alternative path-ways of complement in preterm and small for gestational age infants.Pediatr Res 18:281, 1984.

16. Wolach B, Carmi D, Gilboa S, Satar M, Segal M, Dolfin T, Schle-singer M: Some aspects of the humoral immunity and the phagocyticfunction in newborn infants. Isr J Med Sci 30:331, 1994.

17. Wolach B, Baehner RL, Boxer LA: Review: Clinical and laboratoryapproach to the management of neutrophil dysfunction. Isr J Med Sci18:897, 1982.

18. Harris MC, Shalit M, Southwick FS: Diminished actin polymerizationby neutrophils from newborn infants. Pediatr Res 33:27, 1993.

19. Yamazaki M, Matsuoka T, Yasui K, Komiyama A, Akabane T: In-creased production of superoxide anion by neonatal polymorpho-nuclear leukocytes stimulated with a chemotactic peptide. Am J He-matol 27:169, 1988.

20. Yuli I, Tomonaga A, Snyderman R: Chemoattractant receptor func-tions in human polymorphonuclear leukocytes are divergently alteredby membrane fluidizers. Proc Natl Acad Sci USA 79:5906, 1982.

21. Snyderman R, Yuli I, Koo C: Pharmacological manipulation of thechemotactic factor receptor on leukocytes. In August JT (ed): ‘‘Bio-logical Response Mediators and Modulators.’’ New York: AcademicPress Inc., 1983, p 211.

22. Wolach B, Ben-Dor M, Chomsky O, Gavrieli R, Shinitzky M: Im-proved chemotactic ability of neonatal polymorphonuclear cells in-duced by mild membrane rigidification. J Leukocyte Biol 51:324,1992.

23. Yuli I, Snyderman R: Rapid changes in light scattering from humanpolymorphonuclear leukocytes exposed to chemoattractants. Discreteresponses correlated with chemotactic and secretory functions. J ClinInvest 73:1408, 1984.

24. Boyum A: Isolation of mononuclear cells and granulocytes from hu-man blood. Isolation of mononuclear cells by one centrifugation and ofgranulocytes by combining centrifugation and sedimentation at 1 g.Scand J Clin Lab Invest 21(Suppl 97):77, 1963.

25. Yuli I, Snyderman R: Light scattering by polymorphonuclear leuko-cytes stimulated to aggregate under various pharmacologic conditions.Blood 64:649, 1984.

26. Falk W, Goodwin RH Jr, Leonard EJ: A 48-well microchemotaxisassembly for rapid and accurate measurement of leukocyte migration.J Immunol Methods 33:239, 1980.

27. Clawson CC, Repine JE: Quantitation of maximal bactericidal capac-ity in human neutrophils. J Lab Clin Med 88:316, 1976.

28. Weening RS, Wever R, Roos D: Quantitative aspects of the productionof superoxide radicals by phagocytizing human granulocytes. J LabClin Med 85:245, 1975.

29. Cianciolo GJ, Snyderman R: Monocyte responsiveness to chemotacticstimuli is a property of a subpopulation of cells that can respond tomultiple attractants. J Clin Invest 67:60, 1981.

30. Wallace PJ, Wersto RP, Packman CH, Lichtman MA: Chemotacticpeptide-induced changes in neutrophil actin conformation. J Cell Biol99:1060, 1984.

31. Fechheimer M, Zigmond SH: Changes in cytoskeletal proteins of poly-morphonuclear leukocytes induced by chemotactic peptides. Cell Mo-til 3:349, 1983.

32. Sklar LA, Omann GM, Painter RG: Relationship of actin polimeriza-tion and depolimerization to light scattering in human neutrophils:

14 Wolach et al.

Dependence on receptor occupancy and intracellular Ca2+. J Cell Biol101:1161, 1985.

33. Stossel TP, Chaponnier C, Ezzell RM, et al: Nonmuscle actin-bindingproteins. Annu Rev Cell Biol 1:353, 1985.

34. Cano ML, Lauffenburger DA, Zigmond SH: Kinetic analysis of F-actin depolimerization in polymorphonuclear leukocyte lysates indi-cates that chemoattractant stimulation increases actin filament numberwithout altering the filament length distribution. J Cell Biol 115:677,1991.

35. Sengelov H, Follin P, Kjeldsen L, Lollike K, Dahlgren C, BorregaardN: Mobilization of granules and secretory vesicles during in vivoexudation of human neutrophils. J Immunol 154:4157, 1995.

36. Falloon J, Gallin JI: Neutrophil granules in health and disease. J Al-lergy Clin Immunol 77:653, 1986.

37. Omann GM, Porasik-Lowes MM: Graded G-protein uncoupling bypertussis toxin treatment of human polymorphonuclear leukocytes. JImmunol 146:1303, 1991.

38. Bengtsson T, Sarndahl E, Stendahl O, Andersson T: Involvement ofGTP-binding proteins in actin polimerization in human neutrophils.Proc Natl Acad Sci USA 87:2925, 1990.

39. Ben-Dor M, Ashkenazi M, Wolach B: Bactericidal activity of phago-cytic cells in neonates. Harefuah 114:332, 1988.

40. Miller ME: Developmental maturation of human neutrophil motilityand its relationship to membrane deformability. In Bellanti JA, DaytonDH (eds): ‘‘Phagocytic Cell in Host Resistance.’’ New York: RavenPress, 1975, p 295.

41. Arenson EB, Epstein MB, Seeder RC: Monocyte subsets in neonateand children. Pediatrics 64(Suppl):740, 1979.

42. Klempner MS, Gallin JI: Separation and functional characterization ofhuman neutrophil subpopulations. Blood 51:659, 1978.

43. Mease AD, Burgess DP, Thomas PJ: Irreversible neutrophil aggrega-tion. A mechanism of decreased newborn neutrophil chemotactic re-sponse. Am J Pathol 104:98, 1981.

44. Sklar LA, Oades ZG, Finney DA: Neutrophil degranulation detectedby right angle light scattering: Spectroscopic methods suitable forsimultaneous analyses of degranulation or shape change, elastase re-lease and cell aggregation. J Immunol 133:1483, 1984.

45. Ambruzo DR, Bentwood B, Henson PM, Johnston RB Jr: Oxidativemetabolism of cord blood neutrophils: Relationship to content anddegranulation of cytoplasmic granules. Pediatr Res 18:1148, 1984.

Neonate Neutrophil Inflammatory Response 15